Acute kidney injury (AKI) is a devastating disease with a high mortality rate. Apoptosis and necrosis play major roles in AKI, but there is a fundamental knowledge gap of the factors regulating their induction in AKI. Continued existence of this gap represents a significant problem as AKI has a high mortality rate and there are very few therapeutic interventions to alter the clinical course of this disease. The long-term goal is t uncover mechanisms involved in AKI for the development of novel therapeutics to protect the kidney. Ceramides regulate apoptosis and necrosis and are elevated in the kidney in several models of AKI. The factors that regulate production of ceramides during kidney apoptosis and necrosis and whether ceramides lead to apoptotic versus necrotic kidney cell death are completely unknown. Likewise, there are many different ceramide species and the roles for particular ceramide species in AKI have not been determined. This proposal will answer these questions to achieve the objective of developing ceramides as novel therapeutic approaches for the treatment of AKI. Preliminary data demonstrate that: (i) long-chain ceramides (C16 - C20- ceramides) are generated via de novo synthesis during kidney cell apoptosis and blocking their generation inhibits apoptosis; (ii) the pro-apoptotic BCL-2 protein BAK is a key regulator of ceramide synthases (CerS) and long-chain ceramide generation during kidney cell apoptosis; (iii) acid sphingomyelinase (aSMase) generated very long-chain ceramides (C24-C26-ceramides) occurs in kidney cell necrosis; (iv) kidney cortical CerS and acid SMase are activated and specific ceramides elevated several-fold in in vivo rodent models of AKI (cisplain nephrotoxicity and rhabdomyolysis-induced AKI); and (v) mice given inhibitors of enzymes responsible for ceramide synthesis are protected from cisplatin-induced AKI. This expanding and developing body of work has led us to propose the following hypothesis: nephrotoxic stimuli elevate specific species of kidney ceramides through CerS and SMase-mediated pathways, inducing kidney cell death and ultimately kidney failure. This hypothesis will be tested with three specific aims: (1) determine the mechanism by which BAK regulates CerS activity and generation of specific long-chain ceramides during kidney cell apoptosis; (2) determine the contribution of SMase-generated ceramide to kidney cell necrosis; and (3) determine the in vivo contribution of CerS and aSMase to cisplatin-induced AKI in mice. The approach is innovative because it will identify the mechanism by which BAK regulates ceramide synthases as well as the specific role of individual ceramide species in vivo in AKI. The proposed research is significant as it advances our current knowledge of mechanisms of kidney cell death AKI. Ultimately such knowledge has the potential to greatly improve the treatment of AKI.